During a Dengue virus (DEN) infection, viral-viral and viral-host protein interactions are crucial for the generation of viral progeny and modulation of the host immune response. Disruption of any of these interactions is potentially detrimental to the viral life cycle. Hence, the identification of compounds capable of disrupting these interactions is critically important for the development of effective therapies against DEN infections. We propose to lay a framework for the design, optimization, and testing of small molecule inhibitors for DEN (Fig. 1). We will leverage an ongoing developmental project (started in March 2008) aimed at identifying inhibitory compounds for the viral protease, NS3, by obtaining additional structures with NS3 in association with candidate inhibitors. Lead optimization of candidate compounds will then be performed with efficacy validation being carried out by collaborators in the PSWRCE. We will also conduct structure determination of DEN proteins in complex with known interacting partners. Structural information will then be used to design inhibitory compounds or assays designed to screen for inhibitory compounds. Finally, we will identify additional DEN protein-protein interactions that will serve as targets for co-crystallization and inhibitor design. Specific Aim 1: Continue and extend ongoing structure-based drug design efforts with DEN proteases from all four serotypes. We will perform high-throughput screening assays with screens from the Molecular Libraries Screening Centers Network and commercially available fragment-based screens to identify new lead compounds. We will then obtain co-crystal structures with these compounds to guide rational inhibitor design. Specific Aim 2: Conduct structural studies on identified protein-protein interaction involved in DEN infections. Specific Aim 3: Identify additional pathogen-host protein-protein interactions for structural studies by utilizing affinity purification followed by mass spectrometry analysis to identify interacting partners present in protein complexes isolated from mammalian cells.